The invention relates to a method for determining the copy number of a nucleic acid sequence.
Variation in the copy number of particular nucleic acid sequences, including amplification or deletion, underlies a variety of human disorders. In cancer, amplification of oncogenes or deletion of tumor suppressor genes is found frequently in primary tumors. Currently, detection of variations in the copy number of a particular DNA sequence can be accomplished by one of several methods, such as identifying deletions by loss of heterozygosity (LOH) analyses, and identifying amplifications by Southern blotting analyses.
LOH studies rely on informative sequence variations occurring near the particular DNA sequence of interest and can utilize either polymerase chain reaction (PCR) or Southern blot hybridization analysis to identify the loss of a genomic DNA sequence variant. LOH studies are consequently restricted by the need for polymorphic markers. Particular markers can be uninformative in a given case and can also be located some distance from the DNA sequence of interest.
Other methods, such as quantitative Southern blotting and fluorescence in situ hybridization can detect both deletions and amplifications of a target sequence without the heterozygosity requirements of RFLP or PCR typing methods. However, these methods offer limited resolution, can be technically difficult, labor-intensive, and may require large amounts of material or specialized tissue samples.